The present invention relates to amide derivatives of 1,4-di-substituted piperidines useful in the treatment of cognitive disorders, pharmaceutical compositions containing the compounds, methods of treatment using the compounds, and to the use of said compounds in combination with acetylcholinesterase inhibitors.
Piperidine-derivative muscarinic antagonists useful in the treatment of cognitive disorders such as Alzheimer""s disease are disclosed in U.S. Pat. No. 6,037,352. In particular, U.S. Pat. No. 6,037,352 discloses compounds of the generic formula: 
wherein, inter alia, Y is CH; Z is N; X is xe2x80x94NHCOxe2x80x94; R is substituted benzyl; R1 and R21 are each H; R3, R4, R27 and R28 are hydrogen; and R2 is cycloalkyl. Similar compounds wherein the benzene ring is replaced by a pyridinyl ring are disclosed in U.S. Pat. No. 6,066,636. Compounds of the present invention represent a selection invention over U.S. Pat. Nos. 6,037,352 and 6,066,636.
The present invention relates to compounds of the structural formula I: 
or a pharmaceutically acceptable salt, ester or solvate thereof, wherein
R1 is R5xe2x80x94(C3-C8)cycloalkyl, R5xe2x80x94(C3-C8)cycloalkyl(C1-C6)alkyl, R5-aryl, R5-aryl-(C1-C6)alkyl or R5-heteroaryl;
R2 is H, (C1-C6)alkyl, R6xe2x80x94(C3-C8)cycloalkyl, R6xe2x80x94(C3-C8)cycloalkyl-(C1-C6)alky, R6-heterocycloalkyl, R6xe2x80x94(C6-C10)bridged cycloalkyl, or R6-bridged heterocycloalkyl;
R3 is C1-C6 alkyl or xe2x80x94CH2OH;
R4 is H or C1-C6 alkyl;
R5 is 1-4 substituents independently selected from the group consisting of H, C1-C6 alkyl, halogen, xe2x80x94OH, C1-C6 alkoxy, CF3, xe2x80x94CN, xe2x80x94CO2R4, xe2x80x94CONHR4, xe2x80x94SO2NHR4, xe2x80x94NHSO2R4 and xe2x80x94NHC(O)R4; and
R6 is 1-4 substituents independently selected from the group consisting of H, C1-C6 alkyl, halogen, xe2x80x94OH, C1-C6 alkoxy, CF3, xe2x80x94NH2, (C1-C6)alkylamino, phenyl, C1-C2 alkylenedioxy, and (C1-C6)alkoxycarbonyl.
In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I in a pharmaceutically acceptable carrier. The invention also relates to a method of using a compound of formula I or a pharmaceutical composition comprising a compound of formula I in the treatment of a cognitive disease or neurodegenerative disease comprising administering an effective amount of a compound or composition of this invention to a mammal in need of such treatment.
In still another aspect, the invention relates to a method for treating a cognitive disease or neurodegenerative disease comprising administering to a mammal in need of such treatment an effective amount of a combination of a compound of formula I and an acetylcholinesterase inhibitor.
In a final aspect, the invention relates to a kit for treating a cognitive disease or neurodegenerative disease comprising in separate containers in a single package pharmaceutical compositions for use in combination, in one container a compound of formula I in a pharmaceutically acceptable carrier and in a second container, an acetylcholinesterase inhibitor in a pharmaceutically acceptable carrier, the combined quantities being an effective amount.
Referring to formula I, above, one group of preferred compounds is that wherein R1 is R5-phenyl or R5-cyclohexyl. R5 is preferably H, halogen or C1-C6 alkyl, more preferably H, F or xe2x80x94CH3.
Another group of preferred compounds is that wherein R2 is R6xe2x80x94C3-C8 cycloalkyl, especially R6xe2x80x94C5-C7 cycloalkyl. R6 is preferably H or C1-C6 alkyl.
R3 is preferably xe2x80x94CH3, and R4 is preferably H.
Compared to the compounds specifically disclosed in U.S. Pat. No. 6,037,352 or U.S. Pat. No. 6,066,636, none of which contain the R3 moiety, compounds of the present invention show greater m2 selectivity.
As used herein, the term xe2x80x9calkylxe2x80x9d represents a straight or branched saturated hydrocarbon chain having the designated number of carbon atoms. If the number of carbon atoms is not specified, e.g., if the term lower alkyl is used, chain lengths of 1 to 6 carbons are intended.
xe2x80x9cCycloalkylxe2x80x9d represents a saturated carbocyclic ring having 3 to 8 carbon atoms. Bridged cycloalkyl refers to cycloalkyl rings wherein two non-adjacent ring members are joined by a C1-C2 alkyl chain.
The term xe2x80x9cheterocycloalkyl xe2x80x9d refers to 4- to 7-membered saturated rings comprising 1 to 3 heteroatoms independently selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94and xe2x80x94NR7xe2x80x94, wherein R7 is H or C1-C6 alkyl, and wherein the remaining ring members are carbon. Where a heterocyclic ring comprises more than one heteroatom, no rings are formed where there are adjacent oxygen atoms, adjacent sulfur atoms, or three consecutive heteroatoms. Examples of heterocyclic rings are tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl and piperazinyl. Bridged heterocycloalkyl refers to heterocycloalkyl rings wherein two non-adjacent carbon ring members are joined by a C1-C2 alkyl chain.
Halogen represents fluoro, chloro, bromo or iodo.
Aryl represents phenyl or naphthyl.
Heteroaryl means a 5 or 6-membered aromatic ring comprising 1 to 3 heteroatoms independently selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 and xe2x80x94Nxe2x95x90, provided that the rings do not include adjacent oxygen and/or sulfur atoms. Examples of heteroaryl groups are pyridyl, isoxazolyl, oxadiazolyl, furanyl, pyrrolyl, thienyl, imidazolyl, pyrazolyl, tetrazolyl, thiazolyl, thiadiazolyl, pyrazinyl, pyrimidinyl, pyridazinyl and triazolyl. All positional isomers are contemplated, e.g., 2-pyridyl, 3-pyridyl and 4-pyridyl.
When a variable appears more than once in the structural formula, for example R5, the identity of each variable appearing more than once may be independently selected from the definition for that variable.
Compounds of the invention have at least one asymmetrical carbon atom, i.e., the carbon to which R3 is attached. All isomers, including diastereomers, enantiomers and rotational isomers are contemplated as being part of this invention. The invention includes d and I isomers in both pure form and in admixture, including racemic mixtures. Isomers can be prepared using conventional techniques, either by reacting optically pure or optically enriched starting materials or by separating isomers of a compound of formula I. The preferred stereochemistry of compounds of the invention is shown in formula IA: 
Compounds of formula I can exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms, with pharmaceutically acceptable solvents such as water, ethanol and the like, are equivalent to the unsolvated forms for purposes of this invention.
A compound of formula I may form pharmaceutically acceptable salts with organic and inorganic acids. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic and other mineral and carboxylic acids well known to those skilled in the art. The salts are prepared by contacting the free base forms with a sufficient amount of the desired acid to produce a salt in the conventional manner. The free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous sodium hydroxide, potassium carbonate, ammonia or sodium bicarbonate. The free base forms differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to their respective free base forms for purposes of the invention.
Compounds of formula I can be prepared using methods well known to those skilled in the art, for example by procedures disclosed in U.S. Pat. No. 6,037,352, incorporated herein by reference, or by parallel synthesis or combinatorial chemistry. The skilled artisan will recognize that other procedures may be applicable, and that the procedures may be suitably modified to prepare other compounds within the scope of formula I.
Compounds of formula I as defined above are prepared using a solid phase synthetic procedure as shown in the following Scheme 1, wherein Me is methyl and FMOC is 9-fluorenylmethoxycarbonyl. 
The synthesis in Scheme 1 can be accomplished by the reaction of 9-BBN with an olefin such as 2 followed by the Suzuki coupling with an aryl halide such as 1 to afford compounds 3. Hydrolysis of ester 3 and subsequent removal of the N-Boc provides the amino acid intermediate 5 which is protected by treatment with FmocOSU. This product is then converted into the acid chloride 6 upon treatment with reagents such as POCI3 or oxalyl chloride.
The amine (R1CHR3NH2) is reacted with a resin bound aldehyde such as Argopore-MB-CHO resin (Argonaut Corporation, San Carlos, Calif.) by reductive alkylation with sodium triacetoxyborohydride. Subsequent acylation of the resin bound amine (resin 1) with activated acids such as acid chlorides 7, gives resin 2. Deprotection of the N-Fmoc group, followed by reductive alkylation with aldehydes or ketones, or by reaction with an aldehyde followed by treatment with a Grignard reagent, or by reaction with the appropriate mesylate or alkyl halide, provides a resin bound intermediate which, on treatment with TFA, produces compounds of formula I.
Compounds of formula I are also prepared by conventional synthetic chemistry. For example, compounds of formula la, wherein R1 is R5-phenyl, R3 is xe2x80x94CH3 and R4 is hydrogen are prepared as shown in Scheme 2: 
Reaction of amines such as 8 with activated carboxylic acids such as the acid chloride 9 in the presence of a base such as pyridine or triethylamine yields amides of type 10. Treatment of these with an acid such as TFA or HCl gives compounds 11. The piperidine nitrogen of compounds 11 is derivatized to give compounds of type la by reductive alkylation with either aidehydes or ketones in the presence of a reducing agent such as sodium triacetoxyborohydride, or alternatively by reaction with an aldehyde followed by treatment with a Grignard reagent. Yet another method involves reaction of the amine 11 with the appropriate mesylate or alkyl halide in the presence of base.
Starting materials of formula 7, 8 and 9 are known in the art, or are prepared by method well known in the art, as are the ketones and aldehydes used to introduce R2 via reductive alkylation or alkylation with alkyl halide or tosylates.
The above reaction may be followed if necessary or desired by one or more of the following steps; (a) removing any protective groups from the compound so produced; (b) converting the compound so-produced to a pharmaceutically acceptable salt, ester and/or solvate; (c) converting a compound in accordance with formula I so produced to another compound in accordance with formula I, and (d) isolating a compound of formula I, including separating stereoisomers of formula I.
Based on the foregoing reaction sequence, those skilled in the art will be able to select starting materials needed to produce any compound in accordance with formula I.
The compounds of formula I exhibit selective m2 muscarinic antagonist activity, which has been correlated with pharmaceutical activity for treating cognitive disorders and/or symptoms thereof. Examples of cognitive disorders are Alzheimers disease and senile dementia, with treatment resulting in improvement in memory and learning.
The compounds of formula I display pharmacological activity in test procedures designated to indicate m1 and m2 muscarinic antagonist activity. Following are descriptions of the test procedures.
The compound of interest is tested for its ability to inhibit binding to the cloned human m1, m2, m3, m4 and m5 muscarinic receptor subtypes. The sources of receptors in these studies were membranes from stably transfected CHO cell lines which were expressing each of the receptor subtypes. Following growth, the cells were pelleted and subsequently homogenized using a Polytron in 50 volumes cold 10 mM Na/K phosphate buffer, pH 7.4 (Buffer B). The homogenates were centrifuged at 40,000xc3x97g for 20 minutes at 4xc2x0 C. The resulting supernatants were discarded and the pellets were resuspended in Buffer B at a final concentration of 20 mg wet tissue/ml. These membranes were stored at xe2x88x9280xc2x0 C. until utilized in the binding assays described below.
Binding to the cloned human muscarinic receptors was performed using 3H-quinuclidinyl benzilate (QNB) (Watson et al., 1986). Briefly, membranes (approximately 8, 20, and 14 xcexcg of protein assay for the m1, m2, and m4 containing membranes, respectively) were incubated with 3H-QNB (final concentration of 100-200 pM) and increasing concentrations of unlabeled drug in a final volume of 2 ml at 25xc2x0 C. for 90 minutes. Non-specific binding was assayed in the presence of 1 xcexcM atropine. The incubations were terminated by vacuum filtration over GF/B glass fiber filters using a Skatron filtration apparatus and the filters were washed with cold 10 mM Na/K phosphate butter, pH 7.4. Scintillation cocktail was added to the filters and the vials were incubated overnight. The bound radioligand was quantified in a liquid scintillation counter (50% efficiency). The resulting data were analyzed for IC50 values (i.e. the concentration of compound required to inhibit binding by 50%) using the EBDA computer program (McPherson, 1985). Affinity values (Ki) were then determined using the following formula (Cheng and Prusoff, 1973);       K    i    =            IC      50              1      +              [                              concentration            ⁢                          xe2x80x83                        ⁢            of            ⁢                          xe2x80x83                        ⁢            radioligand                                              affinity              ⁢                              xe2x80x83                            ⁢                              (                                  K                  D                                )                            ⁢                              xe2x80x83                            ⁢              of              ⁢                              xe2x80x83                            ⁢              radioligand                        ⁢                          xe2x80x83                                      ]            
Hence, a lower value of Ki indicates greater binding affinity.
To determine the degree of selectivity of a compound for binding the m2 receptor, the Ki value for m1 receptors was divided by the Ki value for m2 receptors. A higher ratio indicates a greater selectivity for binding the m2 muscarinic receptor.
The following procedure is used to show that a compound functions as an m2 antagonist.
Surgery: For these studies, male Sprague-Dawley Rats (250-350 g) were anesthetized with sodium pentobarbital (54 mg/kg, ip) and placed on a Kopf sterotaxic apparatus. The skull was exposed and drilled through to the dura at a point 0.2 mm anterior and 3.0 mm lateral to the bregma. At these coordinates, a guide cannula was positioned at the outer edge of the dura through the drilled opening, lowered perpendicularly to a depth of 2.5 mm, and permanently secured with dental cement to bone screws. Following the surgery, rats were given ampicillin (40 mg/kg, ip) and individually housed in modified cages. A recovery period of approximately 3 to 7 days was allowed before the microdialysis procedure was undertaken.
Microdialysis: All of the equipment and instrumentation used to conduct in vivo microdialysis was obtained from Bioanalytical Systems, Inc. (BAS). The microdialysis procedure involved the insertion through the guide cannula of a thin, needle-like perfusable probe (CMA/12,3 mmxc3x970.5 mm) to a depth of 3 mm in striatum beyond the end of the guide. The probe was connected beforehand with tubing to a microinjection pump (CMA/100). Rats were collared, tethered, and, following probe insertion, were placed in a large, clear, plexiglass bowl with litter material and access to food and water. The probe was perfused at 2 xcexcl/min with Ringer""s buffer (NaCl 147 mM; KCl 3.0 mM; CaCl2 1.2 mM; MgCl2 1.0 mM) containing 5.5 mM glucose, 0.2 mM L-ascorbate, and 1 xcexcM neostigmine bromide at pH 7.4). To achieve stable baseline readings, microdialysis was allowed to proceed for 90 minutes prior to the collection of fractions. Fractions (20 xcexcl) were obtained at 10 minute intervals over a 3 hour period using a refrigerated collector (CMA/170 or 200). Four to five baseline fractions were collected, following which the drug or combination of drugs to be tested was administered to the animal. Upon completion of the collection, each rat was autopsied to determine accuracy of probe placement.
Acetylcholine (ACh) analysis: The concentration of ACh in collected samples of microdialysate was determined using HPLC/electrochemical detection. Samples were auto-injected (Waters 712 Refrigerated Sample Processor) onto a polymeric analytical HPLC column (BAS, MF-6150) and eluted with 50 mM Na2HPO4, pH 8.5. To prevent bacterial growth, Kathon CG reagent (0.005%) (BAS) was included in the mobile phase. Eluent from the analytical column, containing separated ACh and choline, was then immediately passed through an immobilized enzyme reactor cartridge (BAS, MF-6151) coupled to the column outlet. The reactor contained both acetylcholinesterase and choline oxidase covalently bound to a polymeric backbone. The action of these enzymes on ACh and choline resulted in stoichiometric yields of hydrogen peroxide, which was electrochemically detected using a Waters 460 detector equipped with a platinum electrode at a working potential of 500 mvolts. Data acquisition was carried out using an IBM Model 70 computer equipped with a microchannel IEEE board. Integration and quantification of peaks were accomplished using xe2x80x9cMaximaxe2x80x9d chromatography software (Waters Corporation). Total run time per sample was 11 minutes at a flow rate of 1 ml/min. Retention times for acetylcholine and choline were 6.5 and 7.8 minutes, respectively. To monitor and correct for possible changes in detector sensitivity during chromatography, ACh standards were included at the beginning, middle and end of each sample queue.
Increases in ACh levels are consistent with presynaptic m2 receptor antagonism.
A solution of a compound of formula I at a final substrate concentration of 0.5 mg/ml and human, cynomolgus monkey or rat liver microsomes at final P450 concentrations of 0.18, 0.175 and 0.25 nmol/ml, respectively, is incubated in 0.1 M potassium phosphate buffer at pH 7.4 in 96-well micro-titre plates at 37xc2x0 C. for 3 min in a shaking water bath. A cofactor solution containing MgCl2, Glucose-6-phosphate, NADPH, and Glucose-6-phosphate dehydrogenase is added to each sample (half the total incubation volume/sample) and the total incubation mixture is incubated for 0 and 30 min. (An n=3 samples is incubated for each compound). After each time point, an equal volume of CH3CN is added. The samples are mixed by vortexing and the plates are centrifuged at 3000 rpm for 20 min. The supernatant is analyzed by Liquid Chromatography Mass Spectrometry (LCMS) for parent drug and/or metabolites using an appropriate analytical method.
For the compounds of this invention, the following ranges of muscarinic antagonistic activity were observed:
m1: 20 to 2000 nM, with preferred compounds being between 200-1000 nM m2: 1 to 500 nM, with preferred compounds being  less than 5 nM, more preferably  less than 10 nM.
In the microsomal stability assay, the compound of Example 2 gave the following results (% remaining after 30 min.): ratxe2x80x9479%; monkeyxe2x80x9480%; humanxe2x80x9480%.
In the aspect of the invention relating to a combination of a compound of formula I with an acetylcholinesterase inhibitor, examples of acetylcholinesterase inhibitors are donepezil, heptylphysostigmine, tacrine, rivastigmine and galantamine.
For preparing pharmaceutical compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid.
Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art, e.g. magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington""s Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa.
Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injection or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.
Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g. nitrogen.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.
The compounds of the invention may also be deliverable transdermally. The transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
Preferably the compound is administered orally.
Preferably, the pharmaceutical preparation is in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.
The quantity of active compound in a unit dose of preparation may be varied or adjusted from about 1 mg to about 100 mg, preferably from about 1 mg to about 50 mg, more preferably from about 1 mg to about 25 mg, according to the particular application.
The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required.
The amount and frequency of administration of the compounds of the invention and/or the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated. A typical recommended daily dosage regimen for oral administration can range from about 1 mg/day to about 300 mg/day, preferably 1 mg/day to 50 mg/day, in two to four divided doses.
When a compound of formula I is used in combination with an acetylcholinesterase inhibitor to treat cognitive disorders these two active components may be co-administered simultaneously or sequentially, or a single pharmaceutical composition comprising a compound of formula I and an acetylcholinesterase inhibitor in a pharmaceutically acceptable carrier can be administered. The components of the combination can be administered individually or together in any conventional oral or parenteral dosage form such as capsule, tablet, powder, cachet, suspension, solution, suppository, nasal spray, etc. The dosage of the acetylcholinesterase inhibitor may range from 0.001 to 100 mg/kg body weight.